Elution analysis

Hence, once a B ion is freed from the resin, it is immediately complexed and there is much less tendency for it to be resorbed lower down the column as would happen with a stable cationic species. This is an illustration of separation by elution analysis. Its most important application is in the separation of rare earths. When used on a laboratory scale in chemical analysis, this separation technique is known as ion-exchange chromatography. 

Ferric ion was immobilized on a Chelating Sepharose Fast Flow column preparatory to the separation of seven enkephalin-related phosphopep-tides.17 Non-phosphorylated peptides flowed through the column, and the bound fraction contained the product. The capacity of the column was found to be 23 pmol/mL by frontal elution analysis. Cupric ion was immobilized on Chelating Superose for the isolation of bovine serum albumin.18 Cupric ion was immobilized on a Pharmacia HiTrap column for the separation of Protein C from prothrombin, a separation that was used to model the subsequent apparently successful separation of Factor IX from prothrombin Factor IX activity of the eluate was, however, not checked.19 Imidazole was used as the displacement agent to recover p-galactosidase from unclarified homogenates injected onto a nickel-loaded IMAC column.20 Pretreatment with nucleases and cleaning in place between injections were required procedures. A sixfold purification factor was observed. 

Tiselius, A. Developed liquid chromatography and pointed out frontal analysis, elution analysis, and displacement development. 

Fro. 4.—Elution Analysis, on Carbon, of a Mixture of n-Glucose, Sucrose, and Raffinose Developed with Water and Ethanol-Water Mixtures (Refractive Index rs. Volume of Effluent). 

In order to familiarize himself with macromolecules, he spent a postdoctoral year (1950-1951) with Professor A. Tiselius in Sweden. Together with Aim and Hagdahl they developed the method of gradient elution analysis. On his return to England, he showed how to apply temperature gradients to columns which, with solvent gradients, permit high polymer fractionation. These are called Baker-Williams columns. 

Fig. 24.—Elution analysis of oligosaccharides formed by partial acid hydrolysis of a-dextrin. The last peak at fractions 75-80 consists of unchanged a-dextrin.

Kohn, K. W., and Grimek-Ewig, R. A. (1973). Alkaline elution analysis, a new approach to the study of DNA single-strand intemiptions in cells. Cancer Res 33, 1849-1853. 

Besides elution analysis there are two other procedures of separating and removing the various components of the given mixture from a column these are frontal analysis and displacement analysis . 

Frontal analysis and displacement analysis are of secondary importance only in comparison with elution analysis and neither of these two methods has any advantage over elution, except in preparative-scale separations where quantities to be dealt with are large. 

There are two methods for the chromatographic analysis of the reaction products first, elution analysis, which is used by most workers and provides the basis for all commercial instruments and second, frontal analysis, developed by Rezl and co-workers [12, 24, 25]. The frontal method has a number of advantages over the elution method ... 

Note that direct elution analysis of carbon dioxide even in large samples (25 ml) fails to determine trace hydrocarbons without concentration. The method may be recommended for the trace analysis of other acid gases (e.g., hydrogen sulphide, hydrocyanic acid). A chemical concentration method based on removal of unsaturated gaseous hydrocarbons (main component) for determination of trace amounts of hydrogen and carbon dioxide was developed by Alekseeva and co-workers [56, 57]. For absorption of olefins use was made of a column containing a solution of silver nitrate and mercury salts. 

Under suitable conditions it is possible to obtain separations on an ion exchange column by elution analysis. The pH of the developing solution should be such that the solutes are distributed between the resin and solvent so that they move down the column as definite bands. 

In many cases simple purification by the frontal analysis technique, followed by simple elution, is not satisfactory, since impurity ions would absorb and elute together with the desired ions. It is then necessary to purify the desired ion by elution analysis . An eluting agent is chosen which will displace the various ionic species down the column at different rates. The rate is in fact inversely proportional to the distribution coefficient in each case. To apply the technique successfully, absorption should have taken place mainly in a narrow band at the top of the column, so leaving the lower part free to allow a high degree of separation between the bands of different ions as they are eluted at their respective rates from top to bottom of the column. This is usually ensured by having the column in two sections, only the top one of which is in fact used for absorption. Alternatively, a portion of loose resin may be allowed to absorb the mixture of ions batchwise and then be placed on top of a fresh column. 

When it is necessary to separate cations of close chemical similarity and the technique of elution analysis must be applied, quite different design... 

It is possible to separate zirconium and halfnium by cation-exchange, but they must both be first absorbed upon the resin and then subjected to elution analysis down a column of resin. This process was first described briefly by Street and Seaborg in 1948. They absorbed zirconium and hafnium from the oxychloride solution upon the cation-exchange resin... 

Anion-exchange has also been used for the separation of zirconium and hafnium but it appears less promising for even a small-scale commercial process. This is based upon absorption of zirconium and hafnium complex fluorides upon, for example, Amberlite IRA-40081 or Dowex82 resins, followed by elution analysis with, for example, 0-2 M hydrochloric acid and 0-01 M hydrofluoric acid. i The fluozirconate and fiuohafniate ions were eluted, i.e. [ZrFa] and [HfFe] , the zirconium entering the eluate first. 

Elution analysis corresponds more closely to the normal procedure in chromatographic analysis. A sample of the solution to be analyzed is pressed into the column and is washed through by addition of large quantities of solvent. Fig. 8 shows a diagram of an elution analyris... 

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